Mouse model of retinal degeneration

ABSTRACT

The invention is directed to a method of producing a non-human mammal having one or more pathological characteristics of retinal degeneration and/or age-related macular degeneration. In particular, the invention provides a method of producing a non-human mammal having age-related macular degeneration (AMD). The invention is also directed to non-human animals produced by the methods described herein. Methods of identifying an agent for use in inhibiting one or more pathological characteristics of retinal degeneration and/or AMD is also encompassed by the invention. Also provided is a method of treating AMD in an individual in need thereof comprising, administering to the individual an agent identified herein.

RELATED APPLICATION(S)

This application is a continuation of International Application No.PCT/US2007/009724, which designated the United States and was filed onApr. 23, 2007, published in English, which claims the benefit of U.S.Provisional Application No. 60/794,450, filed on Apr. 24, 2006. Theentire teachings of the above applications are incorporated herein byreference.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by a grant GM 21249(RGS) from the U.S. National Institute of Health. The Government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

The role of oxidatively modified (e.g., by adduction of an oxidativelytruncated lipid) self proteins in autoimmune diseases has not beenstudied. An example of such an autoimmune disease is age related maculardegeneration (AMD). AMD is the leading cause of blindness in the elderlypopulation in developed countries. Over a third of those over the age of75 currently have some form of this disease. Slowing or preventing theprogression of AMD is an urgent public health goal. The role ofinflammation is believed to be one of the crucial first steps that occurearly on in patients that will eventually develop blinding AMD. In theUSA, the prevalence of AMD in Medicare beneficiaries age 65 or olderincreased from 5.0% to 27.1% between 1991 and 1999. Assuming the USpopulation 65-years and older grows as projected to reach 70.3 millionby 2030, AMD cases in this country will soon exceed 20 million. The useof intravitreal steroids as an adjuvant in the treatments of late stageAMD support the role of inflammatory responses in retinal degeneration.Presently, there are no immunosuppressive therapies used to prevent AMD.

Therefore, a better understanding of immune responses in autoimmunediseases, such as AMD, can lead to the development of diagnostic andtherapeutic modalities that can be used early on before irreversibledamage occurs.

SUMMARY OF THE INVENTION

The invention is directed to a method of producing a non-human mammalhaving one or more pathological characteristics of retinal degeneration.The method comprises administering a composition comprising anoxidatively modified protein (e.g., self protein) to a non-human mammal;and maintaining the non-human mammal under conditions in which one ormore pathological characteristics of retinal degeneration develops inthe non-human mammal, thereby producing a non-human mammal having one ormore pathological characteristics of retinal degeneration.

The invention is also directed to a method of producing a non-humanmammal having one or more pathological characteristics of age relatedmacular degeneration (AMD). The method comprises administering acomposition comprising carboxyethylpyrrole (CEP) modified serum albuminto a non-human mammal on day 0, day 10 and day 60. The non-human mammalis maintained under conditions in which one or more pathologicalcharacteristics of AMD develops in the non-human mammal, therebyproducing a non-human mammal having one or more pathologicalcharacteristics of AMD.

Also encompassed by the invention is a method of producing a non-humanmammal having age-related macular degeneration (AMD). The methodcomprises administering a composition comprising carboxyethylpyrrole(CEP) modified serum albumin to a non-human mammal on day 0 and day 10;and maintaining the non-human mammal under conditions in which AMDdevelops in the non-human mammal, thereby producing a non-human mammalhaving AMD.

The invention is also directed to non-human animals produced by themethods described herein.

A method of identifying an agent for use in inhibiting one or morepathological characteristics of retinal degeneration is also encompassedby the invention. The method comprises administering an agent to beassessed to the non-human mammal described herein. Whether one or morepathological characteristics of retinal degeneration is inhibited in thenon-human mammal is determined and compared to a control. If the one ormore pathological characteristics of retinal degeneration is inhibitedin the non-human mammal compared to a control, then the agent can beused to inhibit one or more pathological characteristics of retinaldegeneration.

The invention is also directed to a method of identifying an agent foruse in inhibiting age related macular degeneration (AMD). The methodcomprises administering an agent to be assessed to the non-human mammaldescribed herein. Whether AMD is inhibited in the non-human mammal isdetermined and compared to a control, wherein if AMD is inhibited in thenon-human mammal compared to a control, then the agent can be used toinhibit AMD.

A method of treating AMD in an individual in need thereof comprising,administering to the individual an agent identified herein is alsoprovided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is the chemical structure of CEP-MSA used to immunize mice.

FIG. 1B shows proteins stained with a Gelcode® Blue (Pierce) (left) andby a Western blot (right) prepared using a polyclonal antibody againstCEP. Two preparations of CEP-MSA are shown. Both are highly immunoactiveto the antibody, but show different mobility in the gel. Thepyrrol/protein molar ratios were determined and indicate thatCEP-1=11/1; CEP-2=6.6/1. MSA=mouse serum albumin; MWM=molecular weightmarker.

FIG. 2 shows bar graphs of IFN-γ generation in ELISPOT assays of lymphnode (left) and spleenic T cells (right) from C57BL/6 mice using the 70day immunization protocol.

FIG. 3 is a graph of an ELISA analysis for the presence of anti-CEPantibodies in mice immunized with CEP-MSA (1-4), with complete Freund'sadjuvant alone (5-7) or in naïve mice (8-10).

FIGS. 4A-4J show the cryosectioning and immunohistochemistry analysis ofouter retinal pathology present in mice immunized with CEP-MSA; largearrows indicate several inflammatory cells immediately adjacent to theRPE or in the IPM; large empty vacuoles are also evident (FIGS. 4C-4I),some of which appear to be intracellular (4I); in 4J the RPE hasdegenerated (asterisks); bar in lower right of 4J represents 25 μm.

FIGS. 5A-5D show confocal, phase contrast and merged images of F4/80immunocytochemistry of frozen tissue recovered from a short term(multiple boost) CEP-MSA immunized mouse.

FIGS. 6A-6C show micrographs of retina from mice (FIG. 6A and FIG. 6C)recovered 12 months following immunization with CEP-MSA in completeFreund's adjuvant (FA) followed by a boost 10 days after the initialimmunization with CEP-MSA in incomplete FA; in FIG. 6A, arrows on thelower border of the micrograph indicate the localization of debris-likematerial present below the RPE; FIG. 6B is from a 78 year old humanfemale AMD donor eye containing soft drusen between the RPE and Bruch'smembrane (at the level of the arrow on right of image).

FIGS. 7A-7B show electron microscopy results from the basal side of theRPE at the level of Bruch's membrane.

FIG. 8 is a schematic of carboxyethylpyrrole chemistry; thefragmentation event specified generates HOHA, a seven carbon fragmentthat can only be derived from DHA; HOHA covalently interacts with theepsilon amino group in protein to produce the hapten CEP.

FIG. 9 is a schematic illustrating lipid oxidation in the retina.

DETAILED DESCRIPTION OF THE INVENTION

AMD is a progressive, multifactorial, polygenic disease with poorlyunderstood etiology. Early stages of the disease are typically termed“dry” AMD and associated with the macular accumulation of extracellulardeposits (drusen) below the retinal pigment epithelium (RPE) on Bruch'smembrane of the retina. Geographic atrophy develops in the later stagesof dry AMD and is characterized by macular loss of RPE and photoreceptorcells. Advanced stage disease or “wet” AMD is characterized by choroidalneovascularization. Oxidative damage and vascular factors are consideredimportant insults in the pathogenesis of age related maculardegeneration (AMD). However, recent reports demonstrate that drusen andsurrounding retinal pigmented cells (RPE) contain immunoreactivecomponents of the immune system that activate, complement and induceimmune complex deposition.

Despite the association between the components of drusen and the immunesystem, little is known about the events that trigger the development ofAMD. Described herein is the investigation of whether the development ofan immune response and inflammation to factors in Bruch's membrane andRPE cells is an important step in the development of AMD. The inventionis based, in part, on the discovery that an inflammatory responseagainst a self, oxidatively modified (e.g., by adduction of anoxidatively truncated lipid) protein (e.g., serum albumin) is likely aninitial step in the induction of an autoimmune response that leads tothe development of AMD. Carboxyethylpyrrole (CEP) protein modifications,uniquely generated from oxidation of docosahexaenoate-containing lipidsare more abundant in ocular tissues from AMD than normal donors and areconcentrated in Bruch's membrane, the blood retinal barrier (see FIG.9). The outer segments of the photoreceptors contain high concentrationsof polyunsaturated fatty acids (PUFAs), especially DHA, in the membranesand are exposed to relatively high oxygen tension, close to that foundin arterial blood. The photooxidative environment in the retina and theDHA rich photoreceptor outer segments provide a ready source of reactiveoxygen species for generating oxidative modifications. A high level ofmitochondrial activity required to maintain membrane potentials inphotoreceptor cells is also a likely source of free radicals that canpromote autoxidative fragmentation of DHA-containing membranephospholipids. PUFAs undergo oxidation in the presence of oxygen oroxygen derived radical species, and elevated levels of CEP proteinadducts and CEP autoantibodies are present in AMD plasma (Gu, X. et al.(2003) J. Biol. Chem., 273:42027-42035; U.S. Published Application No.2004/0265924 A1). One example of a CEP modified protein is CEP modifiedserum albumin. Auto-antibodies against CEP-Human Serum Albumin (HSA)have been detected in patients with AMD.

As shown herein, peripheral immunogeneic immunization of a non-humanmammal (e.g., a murine model) with one or more CEP protein adducts(e.g., CEP-mouse serum albumin (CEP-MSA) in complete freund adjuvant(CFA)) can induce an adaptive immune response comprised of an antigenspecific T cell response and the production of anti-CEP-MSAauto-antibodies that results in the destruction of tissue where CEP ishighly localized. It is likely that the retinal photoreceptors, brainand other sites where CEP adducts are present are affected. Presently,there are no in vivo animal models to study the role of early and lateinflammatory responses in retinal degeneration. There is only one modeldescribed in the use of mice that lack MCP-1 ligand or receptor, andthese take 9 months to 1 year to develop disease (Ambati, et al., NatureMedicine 9:1390-1397 (2003)). The other model used to study choroidalneovascularization observed in the late stages of AMD is laser inducedchoroidal damage and the results obtained utilizing this model can beaffected by the wound healing response associated with the laser burn(Dobi, et al., Arch. Ophthalmol., 107:264-269 (1989)). One embodiment ofthe invention described herein is a model of spontaneous retinaldegeneration that results from an immune specific response to CEPadducts present in retinal photoreceptors in a relatively fast fashion.The present invention is also directed to a method of producing a modelof spontaneous retinal degeneration that results from an immune specificresponse to CEP adducts present in retinal photoreceptors in arelatively fast fashion.

Thus, the studies described herein demonstrate that an autoimmuneresponse against oxidatively modified proteins (e.g., self protein;non-self protein) can be generated in vivo. Accordingly, the presentinvention is directed to methods of producing a nonhuman animal model ofan autoimmune response, non-human animals produced by the methods of thepresent invention (e.g., non-human models of autoimmune disease such asAMD) and use of the non-human animals (e.g., to identify agents thatinhibit one or more pathological characteristics of retinal degenerationand/or AMD).

In particular embodiments, the invention provides for methods ofproducing a non-human animal model of an autoimmune response tooxidatively altered self protein. In this embodiment, the methodcomprises administering to (immunizing) a non-human animal with acomposition (first composition) comprising an (one or more) oxidativelymodified self protein, and optionally an adjuvant. The non-human mammalis then administered (challenged with) one or more compositions (e.g., asecond composition, a third composition, a fourth composition, etc.)comprising an oxidatively modified self protein. The non-human animal ismaintained under conditions in which an autoimmune response develops inthe non-human animal. The second, third, fourth etc, composition can bethe same as, similar to, or different from the oxidatively modifiedprotein in the first composition. For example, the second, third, fourthetc, composition can comprise a different oxidatively modified protein(e.g., an oxidatively modified self protein or an oxidatively modifiednon-self protein that differs from the oxidatively modified protein ofthe first composition); and/or optionally, the second, third, fourthetc, composition can comprise an adjuvant that differs from the adjuvantof the first composition. In a particular embodiment, the second, third,fourth etc, composition is the same as, or similar to, the oxidativelymodified protein in the first composition.

As used herein, the term “animal” includes mammals, as well as otheranimals, vertebrate and invertebrate (e.g., birds, fish, reptiles,insects (e.g., Drosophila species), mollusks (e.g., Aplysia). In aparticular embodiment, the animal is a mammal. The terms “mammal” and“mammalian”, as used herein, refer to any vertebrate animal, includingmonotremes, marsupials and placental, that suckle their young and eithergive birth to living young (eutharian or placental mammals) or areegg-laying (metatharian or nonplacental mammals). Examples of mammalianspecies include humans and primates (e.g., monkeys, chimpanzees),rodents (e.g., rats, mice, guinea pigs) and ruminents (e.g., cows, pigs,horses).

As indicated herein the non-human animal is administered multiple dosesof the composition. That is, the animal can be administered a firstcomposition, a second composition, a third composition, a fourthcomposition, a fifth composition, etc. In one embodiment, the non-humanmammal is administered the composition on day 0, day 10 and/or day 60.In another embodiment, the non-human mammal is administered thecomposition on day 0 and/or on day 10. Other combinations ofadministrations can be used and can depend on a variety of factors suchas the oxidatively modified self protein being administered to, and/orthe particular autoimmune response being developed in the animal, whichis apparent to those of skill in the art.

As shown herein an “oxidatively modified protein”, an “oxidativelymodified self protein” or an “oxidatively modified non-self protein” foruse in the methods of the invention includes a carboxyethylpyrrole (CEP)modified protein (also referred to herein as a CEP protein adduct).Carboxyethylpyrrole (CEP), a unique protein modification derived fromthe oxidation of docosahexaenoate (DHA)-containing lipids, was found tobe more abundant in AMD compared to normal ocular tissues (Crabb, J. W.,et al. (2002) Proc Natl Acad Sci USA 99, 14682-7) and was localized inBruch's membrane between the blood-bearing choriocapillaris and RPE.Carboxyethylpyrrole (CEP) protein adducts belong to a family of2-({acute over (ω)}-carboxyalkyl)pyrrole adducts generated from theoxidation of polyunsaturated fatty acids (PUFA) (see Gu et at, J. Biol.Chem., 278(43):42027-42035 (2003) and U.S. Published Application No.2004/0265924, both of which are incorporated herein by reference).Docosahexaenoic acid (DHA) gives rise to 2-({acute over(ω)}-carboxyethyl)pyrrole adducts, by oxidative cleavage to4-hydroxy-7-oxohept-5-enoic acid (HOHA) and reaction of the HOHA withprotein (FIG. 12). HOHA can form an adduct with one or more primaryamino groups of a peptide (e.g., a dipeptide) or protein resulting in aCEP epitope that is referred to as CEP-peptide or CEP-protein adducts,respectively. For example, HOHA can form an adduct with, or on, proteinssuch as albumin, and fragments thereof. CEP epitopes can also begenerated by the reaction of HOHA with the primary amino group ofethanolamine phospholipids that are referred to as ethanolaminephospholipid CEP adducts. Also phospholipids containing an HOHA residuecan form CEPs through reaction with primary amino groups of biomoleculessuch as proteins followed by phospholipolysis of the initially formedCEP phospholipid ester derivative. Thus, an “oxidatively modifiedprotein” for use in the methods of the present invention can alsoinclude other 2-({acute over (ω)}-carboxyalkyl)pyrrole protein adductswhich when administered to an animal, and subsequently used to challengethe immune system of the animal, results in the production of anon-human animal model of an autoimmune response to the oxidativelymodified self protein. For example, other 2-({acute over(ω)}-carboxyalkyl)pyrrole protein adducts include carboxypropylpyrroles,carboxyheptylpyrroles, levuglandin and isolevuglandin (U.S. Pat. No.5,686,250 and U.S. Pat. No. 7,172,874 which are incorporated herein byreference).

Although the invention is described in the exemplification in terms ofan oxidatively modified self protein, it is reasonable to expect that animmune response to CEP epitopes can be generated using any suitableimmunogenic entity that presents a CEP to the immune system of ananimal. That is, the nonhuman animals of the invention can be generatedusing any suitable CEP epitope capable of generating an immune responsethat is directed to a (one or more) CEP-modified self protein. In aparticular embodiment, the nonhuman animals of the invention can begenerated using any suitable CEP epitope capable of generatingantibodies that react (e.g., cross react, specifically bind, aredirected against, have binding specificity for) one or more CEP-modifiedself proteins.

A variety of proteins (polypeptides) which can carry (are capable ofcarrying) a 2-({acute over (ω)}-carboxyalkyl)pyrrole such as CEP can beused in the methods of the present invention. In one embodiment, theprotein is a self protein or portion thereof which forms or carries (iscapable of forming or carrying) a 2-({acute over(ω)}-carboxyalkyl)pyrrole and which generates (is capable of generating)an immune response that is directed to a (one or more) CEP-modified selfprotein. As used herein a “self protein” is any protein present in, ornormally present in, an animal and which can form an adduct with a2-({acute over (ω)}-carboxyalkyl)pyrrole. An example of a self proteinthat can be used in the methods described herein is a serum protein.Specific examples of self proteins which can be used in the methods ofthe invention include albumin, ovalbumin, cystallin, ceruloplasmin,fibronectin, serum amyloid P, actinin, beta B1 and other circulatingprotein and/or peptides. In a particular embodiment, the oxidativemodified self protein is CEP-mouse serum albumin (CEP-MSA).

In another embodiment, the protein is a non-self protein or portionthereof which forms or carries a 2-({acute over(ω)}-carboxyalkyl)pyrrole and generates an immune response that isdirected to a (one or more) CEP-modified self protein. As used herein, a“non-self protein” is any protein that is not normally present in theanimal (a foreign or exogenous protein) and which can form an adductwith a 2-({acute over (ω)}-carboxyalkyl)pyrrole. Non-self proteinsinclude self proteins that have been modified (and thus, as modified arenot normally present in an animal) using methods known to those of skillin the art. A specific example of a self protein which can be used inthe methods of the invention includes keyhole limpet hemocyanin (KLH).

Thus, an oxidatively modified self protein, non-self protein or portionthereof can be modified to incorporate a 2-({acute over(ω)}-carboxyalkyl)pyrrole and administered to the animal for use in themethods of the invention. The portion of the self protein, non-selfprotein or portion thereof can comprise a portion which is capable ofstimulating an immune response either alone or as modified toincorporate a 2-(ÿ-carboxyalkyl)pyrrole in an animal. One or moreportions of the protein (peptide) can be used which can be contiguous ornoncontiguous portions of the protein. For example, the portion can bean antigenic portion (e.g., one or more portions which include one ormore epitopes of a protein) of a self protein.

The amount of a 2-({acute over (ω)}-carboxyalkyl)pyrrole such as CEPadducted to a protein or a portion thereof for use in, the methods ofthe invention typically range from about 5 moles to about 15 moles2-({acute over (ω)}-carboxyalkyl)pyrrole per about 1 mole or about 2moles of protein or portion thereof. In another embodiment, the range isfrom about 6 moles to about 12 moles 2-({acute over(ω)}-carboxyalkyl)pyrrole per mole of protein or portion thereof. Inparticular embodiments, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15moles of a 2-({acute over (ω)}-carboxyalkyl)pyrrole is adducted to about1 mole of a protein or portion thereof.

The dosage of oxidatively modified self protein typically administeredto a non-human animal in the methods of the invention typically rangefrom about 10 μg to about 200 μg, from about 20 μg to about 150 μg, fromabout 30 μg to about 100 μg, and from about 40 μg to about 50 μg. Aswill be apparent to one of skill in the art, the dosage administeredwill depend upon factors such as the period of time over which theoxidatively modified protein is to be administered.

As shown herein the oxidatively modified self protein can beadministered with an adjuvant. Examples of suitable adjuvants that canbe used in the methods include complete Freund's adjuvant (CFA),incomplete Freund's adjuvant (IFA), Bordetella pertussis and alum.

Any variety of methods can be used to administer the oxidativelymodified self protein to the non-human animal. As know to one of skillin the art, the route of administration will depend upon a variety offactors, such as the way in which the oxidatively modified self proteinhas been formulated for delivery. Examples of suitable routes ofadministration include subcutaneous, intravenous, intradermal,intramuscular, intraperitoneal, intraocular, topical, oral andintranasal. Other suitable methods of introduction can also include genetherapy, rechargeable or biodegradable devices, particle accelerationdevises (“gene guns”) and slow release polymeric devices.

2-({acute over (ω)}-carboxyalkyl)pyrrole adducted protein such as CEPadducted protein can be recognized by T cells and an antibody responsecan be elicited after immunization with the CEP adducted protein (e.g.,a CEP adducted serum protein such as CEP-MSA). Moreover, the generationof such an autoimmune response results in tissue damage of retinalphotoreceptors where the CEP antigen is highly localized. The datadescribed herein demonstrate an autoimmune T and B cell response tooxidative modified self proteins that results in tissue specificdisruption. In particular embodiments, the present invention provides amodel in which the development of spontaneous immune mediated retinaldegeneration can be used to study the role of inflammation in autoimmunediseases, such as AMD and other autoimmune diseases.

In one embodiment, the present invention provides a method of producinga non-human mammal having one or more pathological characteristics ofretinal degeneration. The method comprises administering a compositioncomprising an oxidatively modified self protein to a non-human mammal.The non-human mammal is maintained under conditions in which one or morepathological characteristics of retinal degeneration develops in thenon-human mammal, thereby producing a non-human mammal having one ormore pathological characteristics of retinal degeneration.

Pathological characteristics of retinal degeneration include thepresence of anti-2-(ÿ-carboxyalkyl)pyrrole antibodies (e.g., anti-CEPantibodies), lysis of retinal pigmented epithelium (RPE) cells, invadinginflammatory cells in the interphotoreceptor matrix (IPM), focal loss ofRPE, sub-RPE deposits, drusen, complement deposition in Bruch'smembrane, RPE lysis/atrophy, photoreceptor loss, macular degeneration,geographic atrophy, macular edema, diabetic retinopathy or a combinationthereof in the non-human animal. As one of skill in the art willappreciate, a particular combination of pathological characteristics ofretinal degeneration can also lead to age-related macular degeneration(AMD) in the animal.

Thus, the invention also provides a method of producing a non-humanmammal having one or more pathological characteristics of AMD. Themethod comprises administering a composition comprising an oxidativelymodified self protein to a non-human mammal. The non-human mammal ismaintained under conditions in which one or more pathologicalcharacteristics of AMD develops in the non-human mammal, therebyproducing a non-human mammal having one or more pathologicalcharacteristics of AMD.

In another embodiment, the invention provides a method of producing anon-human mammal having AMD. In this embodiment, the method comprisesadministering a composition comprising carboxyethylpyrrole (CEP)modified serum albumin to a non-human mammal on day 0 and day 10. Thenon-human mammal is maintained under conditions in which AMD develops inthe non-human mammal, thereby producing a non-human mammal having AMD.The ADM which develops in the non-human animal can be, for example, dryAMD or wet AMD.

Conditions under which the non-human animals of the invention aremaintained so that pathological characteristics of retinal degenerationand/or AMD develop will be apparent to one of skill in the art andincludes normal animal room illumination (e.g., 12 hours in light/12hours in darkness).

Whether the non-human animal develops pathological characteristics ofretinal degeneration and/or AMD can be determined using skills known inthe art. For example, a fundus examination and/or a histologicalanalysis, can be performed to identify pathological characteristics ofretinal degeneration and/or AMD in the non-human animal. Specifically,focal loss of RPE can be observed in a fundus examination. The loss ofRPE can be evaluated using histology and is evident by the focal absenceof RPE cells resulting in an interrupted RPE with only Bruch's membranepresent.

It is also reasonable to expect that the non-human animals of theinvention can be produced using T cells that are specific for theCEP-modified self protein. That is, T cells that are specific for theCEP-modified self protein are isolated (e.g., purified, substantiallypurified, partially purified) from the non-human animals produced asdescribed herein. The isolated T cells are then administered to anon-human animal that does not have one or more pathologicalcharacteristics of retinal degeneration (a normal, non-human animal).The animal is maintained under conditions in which the T cells attackCEP in the animal which results in the manifestation of one or morepathological characteristics of retinal degeneration in the animal.

The invention also encompasses non-human animals produced by the methodsdescribed herein. In one embodiment, the non-human animal is a non-humananimal model of an autoimmune response to oxidatively altered selfprotein produced by the methods of the present invention (e.g.,non-human models of autoimmune disease). In other embodiments, thenon-human animal has one or more pathological characteristics of retinaldegradation and/or one or more pathological characteristics of AMD. In aparticular embodiment, the non-human animal is a non-human animal modelof AMD.

AMD is the leading cause of blindness in the elderly population indeveloped countries. Over a third of those over the age of 75 currentlyhave some form of this disease. Slowing or preventing the progression ofAMD is an urgent public health goal. The role of inflammation isbelieved to be one of the crucial first steps that occur early on inpatients that will eventually develop blinding AMD. Therefore, theunderstanding of immune responses in autoimmune diseases such as AMDlead to the development of diagnostic and therapeutic modalities thatcan be used early on before irreversible damage occurs. Presently, thereare no in vivo models of immune mediated AMD. Therefore, the presentinvention provides unique models which can be used in a variety ofapplications. Examples of such applications are described below in termsof CEP, however, one of skill in the art will appreciate that theapplication can apply to other 2-({acute over (ω)}-carboxyalkyl)pyrrolesas well as protein modifications by other reactive oxidatively damagedlipids such as levuglandins and isolevuglandins. Thus, the non-humananimal models described herein can be used in methods to identify and/ordevelop; inflammatory systemic signals of early inflammatory responsesin pre-retinal degeneration or other autoimmune diseases associated withanti-CEP response;

inflammatory systemic signals of inflammatory responses during thegeneration of retinal degeneration or other autoimmune diseasesassociated with anti-CEP response;

inflammatory local signals of early inflammatory responses inpre-retinal degeneration or other autoimmune diseases associated withanti-CEP response;

inflammatory local signals of inflammatory responses during thegeneration of retinal degeneration or other autoimmune diseasesassociated with anti-CEP response;

immune related genes associated with increased risk of developingretinal degeneration or other autoimmune diseases associated withanti-CEP response;

inflammatory markers that can be used to monitor systemicimmunosuppression of CEP mediated immune responses;

biological response modifiers to block CEP immune responses in retinaldegenerations and other autoimmune diseases (systemic and local);

pathogenic CEP specific T cell clones that can be used to develop and/orscreen biological response modifiers or pharmaceutical drugs to blockCEP immune responses in retinal degenerations and other autoimmunediseases;

regulatory CEP specific T cell clones that can be used to develop and/orscreen biological response modifiers or pharmaceutical drugs to induceimmune CEP specific protection in retinal degenerations and otherautoimmune diseases;

CEP specific T cell clones that could be used to generate peptides togenerate or induce an immune response (e.g., protective vaccines) toprevent early immune mediated retinal damage and protect patients atrisk of developing AMD and other autoimmune diseases;

the role of other oxidative modifications to a variety of self proteinsand the association of these with the development of AMD and otherautoimmune diseases;

agents (e.g., protein, peptides, oligonucleotides, small molecules) canbe administered to the in vivo models described herein to identify thosethat can be used to treat (e.g., ameliorate the effects of) and/orinhibit (e.g., partially, completely) autoimmune diseases.

Accordingly, the present invention is also directed to a method ofidentifying an agent for use in inhibiting one or more pathologicalcharacteristics of retinal degeneration. The method comprisesadministering an agent to be assessed to the non-human mammal having oneor more pathological characteristics of retinal degeneration anddetermining whether one or more pathological characteristics of retinaldegeneration is inhibited in the non-human mammal compared to a control.If the one or more pathological characteristics of retinal degenerationis inhibited in the non-human mammal compared to a control, then theagent can be used to inhibit one or more pathological characteristics ofretinal degeneration.

The present invention also encompasses a method of identifying an agentfor use in inhibiting AMD. The method comprises administering an agentto be assessed to the non-human mammal having one or more pathologicalcharacteristics of AMD and determining whether age related maculardegeneration is inhibited in the non-human mammal compared to a control.If age related macular degeneration is inhibited in the non-human mammalcompared to a control, then the agent can be used to inhibit age-relatedmacular degeneration.

A suitable control for use in the methods of the invention will beapparent to one of skill in the art. For example, the control can be awild type non-human animal which is the same species as the non-humananimal having one or more pathological characteristics of retinaldegeneration and/or AMD and to which the agent to be assessed has notbeen administered. Alternatively, the control can be a non-human animalhaving one or more pathological characteristics of retinal degenerationand/or AMD to which the agent to be assessed has not been administered.

The terms, “inhibits” and “treat” as used herein, refer not only toameliorating symptoms associated with the condition or disease, but alsopreventing or delaying the onset of the condition or disease, and/orlessening the severity or frequency of symptoms of the condition ordisease.

The agents identified herein can inhibit (partially, completely)activity and/or formation of CEP protein adducts in an individual. Forexample, an agent that inhibits CEP protein adducts can be administeredin order to decrease and/or prevent the activity and/or formation of CEPprotein adducts.

An agent that inhibits a (one or more) CEP protein adduct is an agent orcompound that inhibits the activity and/or formation (expression) of aCEP protein adduct, as described herein (e.g., a CEP protein adductantagonist). An agent that inhibits a CEP protein adduct can alter CEPprotein adduct activity or CEP protein adduct formation by a variety ofmeans. The inhibition can be partial or complete inhibition of CEPprotein adduct activity and/or formation. In addition, the agent caninhibit the CEP protein adduct directly (specifically interact) orindirectly (non-specifically interact).

For example, the agent identified in the methods of the presentinvention can inhibit one or more biological activities of CEP proteinadducts. In one embodiment, the agent binds to all or a portion (e.g., aportion of the CEP protein adduct; the CEP portion of the CEP proteinadduct; the protein or peptide portion of the CEP protein adduct) of theCEP protein adduct under conditions in which the activity of the CEPprotein adduct is inhibited.

Alternatively, the agent identified in the methods of the presentinvention can inhibit formation of the CEP protein adduct. For example,the agent can prevent CEP protein adducts from forming, and/or hydrolyzeCEP protein adducts that have previously formed, regenerating theprimary amino group found in the unmodified biomolecule. The agent canalso interact with the CEP protein adduct or portion thereof after CEPprotein adducts have formed, for example, under conditions in which thepyrrole moiety of the CEP and the protein of the CEP protein adduct isdisrupted. In particular embodiments, the agent cleaves the CEP groupfrom the protein.

Examples of agents which can inhibit receptor-mediated effects of CEPprotein adducts include the following: nucleic acids, fragments orderivatives thereof and vectors comprising such nucleic acids (e.g., anucleic acid molecule, cDNA, and/or RNA); polypeptides; peptidomimetics;fusion proteins or prodrugs thereof; antibodies; ribozymes; aptamers;small molecules; and other compounds that inhibit CEP protein adductactivity and/or formation.

In a particular embodiment, the agent or compound that inhibits CEPprotein adduct activity and/or formation is an antibody (e.g., apolyclonal antibody; a monoclonal antibody). For example, antibodiesthat bind all or a portion of one or more CEP protein adducts and thatinhibit CEP protein adduct activity can be used in the methods describedherein (Gu et al., J. Biol. Chem., 278(43):42027-42035 (2003) and U.S.Application No. 2004/0265924, both of which are incorporated herein byreference). In a particular embodiment, the antibody is a purifiedantibody. The term “purified antibody” as used herein refers toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site that selectively binds all or a portion (e.g., abiologically active portion) of a CEP protein adduct. A molecule thatselectively binds to a CEP protein adduct is a molecule that binds to aCEP protein adduct or a fragment thereof, but does not substantiallybind other molecules in a sample (e.g., a biological sample thatnaturally contains the CEP protein adduct). Preferably the antibody isat least 60%, by weight, free from proteins and naturally occurringorganic molecules with which it naturally associated. More preferably,the antibody preparation is at least 75% or 90%, and most preferably,99%, by weight, antibody. Examples of immunologically active portions ofimmunoglobulin molecules include F(ab) and F(ab′)₂ fragments that can begenerated by treating the antibody with enzymes such as pepsin orpapsain, and single chain FV (scFV) fragments.

The term “monoclonal antibody” or “monoclonal antibody composition,” asused herein, refers to a population of antibody molecules that containonly one species of an antigen binding site capable of immunoreactingwith a particular epitope of a CEP protein adduct of the invention. Amonoclonal antibody composition thus typically displays a single bindingaffinity for a particular CEP protein adduct of the invention with whichit immunoreacts.

Polyclonal antibodies can be prepared using known techniques such as byimmunizing a suitable subject with a desired immunogen, e.g., a CEPprotein adduct or fragment thereof. The antibody titer in the immunizedsubject can be monitored over time by standard techniques, such as withan enzyme linked immunosorbent assay (ELISA) using immobilizedpolypeptide. If desired, the antibody molecules directed against the CEPprotein adduct can be isolated from the mammal (e.g., from tissue,blood) and further purified by well-known techniques, such as protein Achromatography to obtain the IgG fraction.

Additionally, recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions,which can be made using standard recombinant DNA techniques, are withinthe scope of the invention. Such chimeric and humanized monoclonalantibodies can be produced by recombinant DNA techniques known in theart.

The present invention is also directed to a method of treatingage-related macular degeneration in an animal in need thereofcomprising, administering to the individual an agent identified in themethods described herein.

The agents which inhibit CEP protein adducts are administered in atherapeutically effective amount (i.e., an amount that is sufficient totreat or inhibit the disease or condition, such as by amelioratingsymptoms associated with the disease or condition, preventing ordelaying the onset of the disease or condition, and/or also lesseningthe severity or frequency of symptoms of the disease or condition). Theamount that will be therapeutically effective in the treatment of aparticular individual's disorder or condition will depend on thesymptoms and severity of the disease, and can be determined by standardclinical techniques. In addition, in vitro or in vivo assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of a practitioner andeach patient's circumstances. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

The methods of the present invention can be used to treat any suitableanimal (individual). In one embodiment, the animal is a primate. In aparticular embodiment, the individual is a human.

The agents (e.g., therapeutic compound) can be delivered in acomposition, as described above, or by themselves. They can beadministered systemically, or can be targeted to a particular tissue.The therapeutic compounds can be produced by a variety of means,including chemical synthesis; recombinant production; in vivo production(e.g., a transgenic animal, such as U.S. Pat. No. 4,873,316 to Meade etal.), for example, and can be isolated using standard means such asthose described herein. A combination of any of the above methods oftreatment can also be used.

The compounds for use in the methods described herein can be formulatedwith a physiologically acceptable carrier or excipient to prepare apharmaceutical composition. The carrier and composition can be sterile.The formulation should suit the mode of administration.

Suitable pharmaceutically acceptable carriers include but are notlimited to water, salt solutions (e.g., NaCl), saline, buffered saline,alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzylalcohols, polyethylene glycols, gelatin, carbohydrates such as lactose,amylose or starch, dextrose, magnesium stearate, talc, silicic acid,viscous paraffin, perfume oil, fatty acid esters,hydroxymethylcellulose, polyvinyl pyrolidone, etc., as well ascombinations thereof. The pharmaceutical preparations can, if desired,be mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, coloring, flavoring and/or aromatic substances andthe like that do not deleteriously react with the active compounds.

The composition, if desired, can also contain minor amounts of wettingor emulsifying agents, or pH buffering agents. The composition can be aliquid solution, suspension, emulsion, tablet, pill, capsule, sustainedrelease formulation, or powder. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,polyvinyl pyrollidone, sodium saccharine, cellulose, magnesiumcarbonate, etc.

Methods of introduction of these compositions include, but are notlimited to, intradermal, intramuscular, intraperitoneal, intraocular,intravenous, subcutaneous, topical, oral and intranasal. Other suitablemethods of introduction can also include gene therapy, rechargeable orbiodegradable devices, particle acceleration devises (“gene guns”) andslow release polymeric devices. The pharmaceutical compositions of thisinvention can also be administered as part of a combinatorial therapywith other compounds.

The composition can be formulated in accordance with the routineprocedures as a pharmaceutical composition adapted for administration tohuman beings. For example, compositions for intravenous administrationtypically are solutions in sterile isotonic aqueous buffer. Wherenecessary, the composition may also include a solubilizing agent and alocal anesthetic to ease pain at the site of the injection. Generally,the ingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container such as an ampule orsachette indicating the quantity of active compound. Where thecomposition is to be administered by infusion, it can be dispensed withan infusion bottle containing sterile pharmaceutical grade water, salineor dextrose/water. Where the composition is administered by injection,an ampule of sterile water for injection or saline can be provided sothat the ingredients may be mixed prior to administration.

For topical application, nonsprayable forms, viscous to semi-solid orsolid forms comprising a carrier compatible with topical application andhaving a dynamic viscosity preferably greater than water, can beemployed. Suitable formulations include but are not limited tosolutions, suspensions, emulsions, creams, ointments, powders, enemas,lotions, sols, liniments, salves, aerosols, etc., that are, if desired,sterilized or mixed with auxiliary agents, e.g., preservatives,stabilizers, wetting agents, buffers or salts for influencing osmoticpressure, etc. The compound may be incorporated into a cosmeticformulation. For topical application, also suitable are sprayableaerosol preparations wherein the active ingredient, preferably incombination with a solid or liquid inert carrier material, is packagedin a squeeze bottle or in admixture with a pressurized volatile,normally gaseous propellant, e.g., pressurized air.

Compounds described herein can be formulated as neutral or salt forms.Pharmaceutically acceptable salts include those formed with free aminogroups such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with free carboxyl groupssuch as those derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

In another embodiment, the invention is directed to agents that inhibitCEP protein adducts for use as a medicament in therapy. For example, theagents identified herein can be used in the treatment of optic nervedamage. In addition, the agents identified herein can be used in themanufacture of a medicament for the treatment of AMD.

Exemplification CEP Immunization and the Development of an AMD-LikePathology in the Mouse

A. Identification of an Inflammatory Signal from the Outer RetinaCausing Age-Related Macular Degeneration.

Purpose: To experimentally demonstrate the linkage between oxidativemodifications of proteins in the outer retina, their recognition by theimmune system and the vulnerability of the outer retina to immune attackleading to age-related macular degeneration (AMD). AMD eye tissuescontain high levels of proteins chemically modified by the adduction ofoxidation fragments from the long chain polyunsaturated fatty acid,docosahexaenoic acid (DHA). Many proteins modified by these and otheradducts are found in drusen and Bruch's membrane. Additionally,autoantibodies against a unique carboxyethylpyrrole (CEP) adduct thatcan only be generated from DHA are more abundant in the circulation(plasma) of individuals with AMD than in age-matched individuals withoutAMD.

Methods: Normal mice were immunized with CEP-adducted mouse serumalbumin. The prediction was that systemic immunization with CEP wouldsensitize mice to endogenously generated CEP-adducts produced in theouter retina during the normal course of aging. In turn the immunesystem would respond by attacking the cells where CEP epitopes are mostreadily generated. Two immunization protocols were used: one withmultiple boosts over a three month period, and the other with a singleboost and maintenance of the mice for up to one year post immunization.Eye tissues were recovered and analyzed with microscopy.

Results: In multiple boost mice dramatic lesions of the RPE involvinglysis of individual cells are evident along with the invasion ofmacrophages and debris removal. In single boost animals focal drusendeposits in the fundus were present along with some RPE loss. None ofthese changes were observed in normal mice immunized with non-adductedmouse serum albumin or when CEP-MSA was used to immunize rag−/−mice thatwere missing mature T cells and B cells.

Conclusions: Mice immunized with CEP-mouse serum albumin developedchanges in the outer retina that are characteristic of those present inhumans with AMD. The high concentration of DHA in the photoreceptors-RPEcomplex coupled and the vulnerability of DHA to oxidative damageresulted in the slow generation of CEP-adducts over time in the outerretina. Normally these CEP-adducts represent new epitopes foreign to theimmune system. When mice were immunized with CEP-mouse serum albuminthey became sensitized to the CEP-adduct and responded with an immuneattack on the cellular source of this adduct the outer retina resultingin these AMD-like changes. This model for AMD in the mouse is animportant a new resource for preclinical testing of therapeuticsdesigned to prevent or limit the progression of AMD.

B. Immune Responses to Oxidatively Altered Self Proteins in the RetinaLead to the Development of Retinal Degeneration

Purpose: Patients with Age Related Macular Degeneration (AMD) havecirculating auto-antibodies to carboxyethylpyrrole (CEP) modifiedalbumin. CEP adducts are generated in the retinal photoreceptors and RPEin response to oxidative stress. The main goal of this experiment was todetermine if the immunization of mice with CEP-modified albumin leads tothe generation of an immune response that induces AMD.

Methods: C57BL/6 and Balb/c mice were immunized in the footpad withmouse serum albumin adducted with CEP (CEP-MSA) or non-adducted MSAemulsified in complete Freund's adjuvant (CFA) at day 0. One group ofmice was followed for a year and a second group of mice was challengedsubcutaneously at day 10 with incomplete Freund's adjuvant-CEP-MSA,followed with a second challenged two months later with CFA-MSA.Clinical examinations of the fundus and electroretinograms (ERG) wereperformed at different post-immunization times and the appearance ofretinal changes were scored. Sera from immunized mice were tested foranti-CEP antibody by ELISA. Mice were sacrificed at different timepoints for histological examination of enucleated eyes and analysis of Tcell priming to CEP in lymph nodes and spleens using Interferon-gamma(IFN-γ) ELISPOT in response to in vitro CEP-MSA stimulation. Rag KO micewere also immunized and challenged.

Results: Mice immunized once developed atrophic retinal changes of theretinal pigmented epithelium (RPE) at 11 months and histologicalexamination showed vacuolization of RPE and patchy areas of loss ofphotoreceptors consistent with dry AMD. Anti-CEP antibodies were alsopresent at this time point and CEP-MSA specific T cells were readilydetected. Mice that were immunized and challenged had increased titersof anti-CEP antibodies at 20 days and detectable CEP-MSA IFN-γ specificT cells in lymph nodes and spleen. Although clinical examination wasunremarkable, histological analysis showed significant amount ofphotoreceptor loss and scattered vacuolization of the RPE. No ERGchanges were noted.

Conclusion: Immune responses to a CEP adducted self-protein in theretina can lead to the generation of an adaptive immune response thatresults in retinal damage consistent with dry AMD. This demonstrates forthe first time that an in vivo chemical modification induced byoxidative stress in a specific organ can lead to autoimmunity. Moreover,this represents an animal model to study dry AMD and develop noveltherapies.

Discussion

Higher levels of CEP-adducted proteins are present in AMD donor eyes andCEP adducted proteins are present in the blood of AMD patients (Crabb,J. et al., Proc Natl Acad Sci (USA) 99:14682-14687 (2002); Hollyfield,J. G., et al., Adv. Exp. Med. Biol. 533:83-89 (2003); Gu, X. et al., JBiol Chem 278:42027-42035 (2003)). As described herein, whether animmune response to CEP adducted self proteins generated in the retinainitiated the development of pathological features associated with AMDwas tested. For the antigen, mouse serum albumin (MSA) was chemicallymodified to generate MSA-CEP (FIGS. 5A-5B) (Gu, X. et al., J Biol Chem278:42027-42035 (2003)). Aliquots of MSA-CEP were emulsified in completeFreund's adjuvant and C57BL/6 and BALB/c mice were immunized following apreviously described immunization regimen (Percopo, C. M., et al., JImmunol 145:4101-4107 (1990)).

Two immunization protocols in the development of this mouse model forAMD were used. A short term immunization protocol with two boostsdesigned as follows:

(A) The short term protocol: Following immunization at day 0 and boostswith incomplete Freund's adjuvant on day 10 and day 60, animals werekilled on day 70 and eyes, spleen, lymph nodes and blood were taken forimmunological and histological analysis. Control immunizations were withcomplete Freund's adjuvant alone followed by incomplete Freund'sadjuvant at the same intervals as indicated above. Tissues of naïve micewere also used as controls.

(B) The long term protocol: Following immunization on day 0 and a singleboost with incomplete Freunds adjuvant on day 10 the mice weremaintained for up to one year before being killed, with eyes, blood,spleen and lymph nodes harvested for analysis as described above.

The reasoning in pursuing these two protocols was based on thehypothesis that multiple immunizations would cause a quicker responseand possibly involve an attack on the cells in the retina whereCEP-adducts were most abundantly generated. In contrast the long term,single boost protocol might result in a slower immune attack on theouter retina with the development of lesions resembling those found inthe human AMD condition.

CEP-ELISPOT Assays, T Cell Priming in CEP-Immunized Mice

Standard immunological assays were used to determine whether T cellpriming to CEP-MSA had occurred, a phenomenon necessary for T celldependent B cell response (Parker, D. C., Ann Rev Immunol 11:331-360(1993)). Spleen and lymph nodes were harvested, their cells cultured andtested for the generation of gamma-interferon (IFN-γ) production inresponse to CEP stimulation using the well established ELISPOT assay. Tcells from these tissues with a memory of the CEP antigen respond inculture to stimulation with CEP-MSA by, the synthesis of IFN-γ. A colorreaction in the cells that produce IFN-γ can be resolved and the numberof responding cells can be counted. T cells from naïve mice and miceimmunized with native MSA that was not adducted with CEP did notrespond. Furthermore cultures stimulated with native MSA did not secreteIFN-γ.

The graphs in FIG. 2 show IFN-γ generation in ELISPOT assays of lymphnode (left) and spleenic T Cells (right) from C57BL/6J mice using the 70day immunization protocol described in the above paragraph. Animals 1-4were immunized with 200 μg MSA-CEP in complete Freund's adjuvant;animals 5-7 were immunized with complete Freund's adjuvant alone; andanimals 8-10 were naive mice. Note that the spleen cells and lymph nodeT cells in 1-4 responded to CEP-MSA stimulation and cells in 5-10 didnot respond.

Anti-CEP Antibody Production in CEP-MSA Immunized Mice

To determine if the CEP-specific T cell responses demonstrated in thesemice promoted B cells to produce antibodies against CEP, sera for CEPantibodies were analyzed by ELISA. These assays (FIG. 3) showedcirculating anti-CEP antibodies at dilutions as low as 1:500 in theimmunized mice, whereas no CEP antibodies were detected in miceimmunized with Freund's adjuvant only or in naïve mice. Antibody titerswere followed in over 120 mice used in these studies and a range of 2-3fold variability in the antibody titer was found between animals thatare immunized using the same antigen and boost protocols.Non-adducted-MSA immunizations were included as an additional control(not shown).

The graph in FIG. 3 shows the results of the ELISA analysis for thepresence of anti-CEP antibodies in mice immunized with CEP-MSA (1-4),with complete Freund's adjuvant alone (5-7) or in naïve mice (8-10).Note the robust antibody titer (high absorbance levels) at the serumdilutions indicated in 1-4, but the absence of an antibody signal inanimals 5-10.

Outer Retina Pathology in the Short Term, Multiple Boost CEP-MSAImmunized Mice

At the time of harvesting, one eye was frozen in OTC (optic tissuecompound, O.C.T., Tissue Tek®) for cryosectioning andimmuno-histochemistry. The other eye was fixed in mixed aldehydes andembedded in plastic for detailed microscopic analysis as describedpreviously (Besharse, J. C. & Hollyfield, J. G., Invest. Ophthalmol.Vis. Sci. 18:1019-1024 (1979)). The lesions noted involved the RPE,initially with changes in the distribution of melanin and loss ofcytoplasmic staining the cytoplasm (FIG. 4A). In other areas putativeinflammatory cells sometimes containing melanin were present adjacent tothe RPE in the interphotoreceptor matrix (FIGS. 4B-4I at arrows).Occasional small vesicles could be observed within individual RPE cells(FIGS. 4B-4C), whereas in other areas extensive vesiculation of theentire RPE cell could be observed and in some areas inflammatory cellswere noted in close proximity to the RPE lesions. More extensivedegeneration was occasionally observed that involved local areas wherethe entire RPE was missing, producing a condition that was similar togeographic atrophy (FIG. 4J). Photoreceptors under areas of RPE atrophyremained but were highly degenerate in appearance. Some variability wasnoted in the pathology present in the 35 mice used in these studies.Some have no pathology while others have multiple lesions in the sameeye.

Identification of Inflammatory Cells that Invade the InterphotereceptorMatrix (IPM) in CEP-MSA Immunized Mice

The identification of the invading cells that move into the outer retinaand are associated with the RPE lesions have been examined with a numberof specific inflammatory cell markers. Using a marker for macrophages(F4/80 from eBiosciences, San Diego) it was shown that some of the cellsthat move into the outer retina are indeed macrophages since they aredecorated with the F4/80 antibody (FIGS. 5A-5D).

In FIGS. 5A-5D, confocal, phase contrast and merged images of F4/80immunocytochemistry of frozen tissue recovered from a short term(multiple boost) CEP-MSA immunized mouse are shown. The images presentedare from the same animal as the fixed tissue image presented in FIG. 4G.FIG. 5A is a low magnification confocal image showing three F4/80positive cells. FIG. 5B is a phase contrast image showing thelocalization of these cells in the outer retina (arrow shows location ofRPE/choroid). FIG. 5C is a merged image of 5A and 5B. It should be notedthat the retinal structure is not maintained well when the mouse eye isfrozen. FIG. 5D is a higher magnification of the three F4/80 positivemacrophages. The cell on the left has uniformly staining cytoplasmwhereas the two on the right have vesicles in the cytoplasm that areunstained with the antibody. Some of these profiles appeared to bemelanin, similar to what was observed in many of the images presented inFIGS. 4A-4J.

Interpretation of the Data from the Short-Term Immunization Protocol

The above data show that CEP-MSA immunized C57BL/6 mice develop CD4 Thelper cell responses to CEP necessary for the elaboration of anti-CEPantibodies. These antibodies may be involved in the deposition of C3 inBruch's membrane and the choroid of the immunized mice. In the retina,RPE lysis and cell death are closely associated with a small number ofinflammatory cells in the IPM (subretinal space). Some of theseinflammatory cells are identified as macrophages based on their labelingwith the macrophage marker F4/80 antibody. The focal loss of RPEresembles a condition of AMD referred to as geographic atrophy, the endstage of the dry form of AMD. The retinal cell type targeted by theimmune system in this short-term, multiple boost immunization protocolis the RPE, which consistently shows focal changes in multiple sites inthe fundus of the eye.

Outer Retina Pathology in the Long-term, Single Boost CEP-MSA ImmunizedMice

Eighteen (18) mice from the long-term protocol that were maintained for12 months following immunization with CEP-MSA were examined. Serumsamples from each of the animals contained a high titer of anti-CEPantibodies, indicating the successful immunization with MSA-CEP 12months earlier. Eye tissues and blood from naïve mice, mice that wereimmunized with non-CEP adducted MSA (normal MSA) and mice immunized withonly Freund's complete adjuvant were also recovered. No evidence foranti-CEP antibodies was found in the serum from these control mice.Detailed histology was performed on all of these samples and electronmicroscopy on representative tissues. In all the CEP-MSA immunized micemany features of dry AMD present in the outer retina were observed.Focal areas in the fundus contain sub-RPE deposits that resemble bothsoft drusen (FIG. 6A) when compared to soft drusen in human AMD tissuesamples (FIG. 6B). Also observed are focal deposits that elevate the RPEand resemble hard drusen (FIG. 6C).

FIGS. 6A-6C show micrographs of retina from mice (FIG. 6A and FIG. 6C)recovered 12 months following immunization with CEP-MSA in completeFreunds adjuvant (FA) followed by a boost 10 days after the initialimmunization with CEP-MSA in incomplete FA. In FIG. 6A, arrows on thelower border of the micrograph indicate the localization of debris-likematerial present below the RPE. FIG. 6B is from a 78 year old humanfemale AMD donor eye containing soft drusen between the RPE and Bruch'smembrane (at the level of the arrow on right of image). Compare thesub-RPE material in FIG. 6A with that in FIG. 6B. In FIG. 6C the RPE iselevated over a focal deposit resembling hard drusen that containsvesicular material (arrows). Because of the highly localized position ofthis material, it resembles hard drusen in human AMD tissues.

Electron microscopy of these sub-RPE deposits show that much of thismaterial consists of membrane and cytoplasmic debris that may haveoriginated from the basal side of the RPE. When these sub-RPE depositswere observed, the basal infoldings of the RPE were absent and the innercollagenous layer of Bruch's membrane and the basal lamina of the RPEcould not be distinguished (FIGS. 7A-7B). None of these changes wereobserved in the control mice. The data presented clearly indicate thatthe initial hypothesis is correct: mice immunized with CEP adducted to aself protein, MSA, will be sensitized to CEP adducts generated in theouter retina where the parent molecule, docosahexaenoic acid (DHA), ispresent in high concentrations and this tissue interface of the outerretina will undergo pathological changes similar to those that occur inAMD.

FIGS. 7A-7B show electron microscopy (EM) results from the basal side ofthe RPE at the level of Bruch's membrane. FIG. 7A shows sub-RPE material(asterisks) shows high electron density deposits surrounded by electronlucent areas suggesting edema. FIG. 7B is from a control mouse showingthe normal appearance of the basal infoldings of the RPE (asterisks).Dashed lines in both figures represent the level of the central elastinlamina of Bruch's membrane. In FIG. 7A, Bruch's membrane is greatlyswollen when compared to the control in 7B, and lamina proximal to theRPE are absent. FIGS. 7A and 7B are presented at the identicalmagnifications.

Interpretation of the Data from the Long-Term, Single Boost CEP-MSAImmunization Protocol

The long-term immunization protocol clearly shows the development ofsub-RPE deposits that resemble soft drusen present in AMD in humans.Normally the high concentration of DHA in the photoreceptors-RPE complexcoupled with the vulnerability of DHA to oxidative damage results in theslow generation of CEP-adducts over time in the outer retina. TheseCEP-adducts represent new epitopes foreign to the immune system and maycause a low grade inflammation. When mice are immunized with CEP-mouseserum albumin using the multiple boost protocol they become sensitizedto the CEP-adduct and respond with an immune attack on the cellularsource of this adduct the outer retina resulting in these AMD-likechanges. In the single boost protocol, a slower inflammation proceedsaccompanied by drusen accumulation below the RPE that is strikinglysimilar to deposits present in dry AMD in humans. Using CEP-MSA, a modelfor the human AMD disease has been created. This mouse model for AMD isan important new resource for preclinical testing of therapeuticsdesigned to prevent or limit the progression of AMD.

The teachings of all patents, published applications and referencescited herein are incorporated by reference in their entirety.

While this invention has been particularly shown and described withreferences to example embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method of producing a non-human mammal having one or morepathological characteristics of retinal degeneration comprising, a)administering a composition comprising an oxidatively modified proteinto a non-human mammal; and b) maintaining the non-human mammal underconditions in which one or more pathological characteristics of retinaldegeneration develops in the non-human mammal, thereby producing anon-human mammal having one or more pathological characteristics ofretinal degeneration. 2.-24. (canceled)